1. Field
This disclosure relates to methods of treating haemostatic disorders.
2. Description of the Related Art
Haemostasis refers to the process of stopping blood loss from a damaged blood vessel. Coagulation, or the formation of blood clots, is an important part of haemostasis. To stop bleeding from a wound, a damaged blood vessel wall is covered by a clot containing platelets and fibrin. Disorders of coagulation can lead to an increased risk of bleeding (hemorrhage) or clotting (thrombosis).
Massive bleeding or transfusion in patients is commonly associated with impaired coagulation. This impaired coagulation can be caused by dilutional coagulopathy, defined as increased bleeding tendency due to dilution of the blood. Fluid compensation is a conventional treatment of extensive blood loss to normalize blood pressure and avoid circulatory shock. However, fluid compensation dilutes the coagulation factors in the remaining blood and impairs their function, which can result in increased bleeding that might be life-threatening.
The in vivo biochemical pathways leading to coagulation are complex and require a large number of factors and cofactors. There are ten factors designated by Roman numerals I-XIII with III, IV and VI being unused designators. Numerous related factors and cofactors modulate the activity of the biochemical pathway. For example, in the tissue factor pathway, following damage to he blood vessel, factor VII (abbreviated FVII as is the convention for the Roman numeral denominated factors) comes into contact with tissue factor (TF) expressed on tissue-factor-bearing cells (stromal fibroblasts and leukocytes). This forms an activated complex (TF-FVIIa), TF-FVIIa activates FIX and FX. FVII is itself activated by thrombin, FXIa, plasmin, FXII, and FXa. The conversion of FX to activated FXa by TF-FVIIa is almost immediately inhibited by tissue factor pathway inhibitor (TFPI). FXa and its co-factor FVa form the prothrombinase complex, which activates pro-throbin to thrombin. Thrombin then activates other components of the coagulation cascade, including FV and FVIII (which activates FXI, which, in turn, activates FIX), and activates and releases FVIII from being bound to von Willebrand factor (vWf). FVIIIa is the co-factor of FIXa, and together they form the “tenase” complex, which activates FX to positively feed back into the cycle. Activation of the pathway produces a burst of activated thrombin. The thrombin activated in this cycle can convert fibrinogen to fibrin which is the protein which forms a clot together with platelets.
Prothrombin (FII) is produced in the liver and is post-translationally modified in a vitamin K-dependent reaction that converts ten glutamic acids on prothrombin to gamma carboxy glutamic acid. Deficiency of vitamin K or administration. of the anticoagulant warfarin inhibits the conversion of Factor II glutamic acid residue to Gla, slowing the activation of the coagulation cascade. Thrombin (FIIa) is produced by enzymatic cleavage of two sites on FII by activated Factor X (FXa). The activity of FXa is greatly enhanced by binding to activated Factor V (FVa). In human adults the normal blood level of prothrombin activity has been measured to be around 1.1 units/ml. Newborn plasma levels of prothrombin steadily increase after birth to reach normal adult levels, from a level of around 0.5 units/ml one day after birth, to a level of around 0.9 units/ml after 6 months of life,
Thrombin, which is formed from prothrombin, has many effects in the coagulation cascade. It is a serine protease that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions. Al Dieri et al. investigated the relationship between clotting factor concentrations, parameters of thrombin generation, and the amount of blood loss in patients with various congenital coagulation factor deficiencies. The authors demonstrated that bleeding tendency was directly associated with the amount of thrombin generation, which varied linearly to the FII concentrations. Al Dieri et al. Thromb Haemost 2002, 88:576-82. Fenger-Erikson et al. also showed, in the case of dilutional coagulopathy, that aside from fibrinogen, coagulation factors including FII, FX , and FXIII also decrease below the expected level following a 32% dilution with hydroxyethyl starch solution. Fenger-Eriksen et al, J Thromb Haemost 2009, 7:1099-105.
Fibrinogen (Factor I or FI) is a soluble plasma glycoprotein that is synthesized by the liver and converted by thrombin into fibrin during blood coagulation. Processes in the coagulation cascade convert pro-thrombin to the activated serine protease thrombin, which converts fibrinogen into fibrin. Fibrin is then cross-linked by factor XIII to form a clot. The normal concentration of fibrinogen in blood plasma is 1.5-4.0 g/L or about 7 μM. In the event of marked blood loss, fibrinogen more than any other procoagulant factors or platelets reaches critically low plasma concentrations. Hiippala et al. Anesth Analg 1995, 81:360-5; Singbartl et al. Anesth Analg 2003, 96:929-35; McLoughlin et al. Anesth Analg 1996, 83:459-65. Small quantities of colloids (less than 1000 ml) can impair fibrin polymerization and thus clot strength. Innerhofer P et al., Anesth Analg 2002, 95:858-65; Mittermayr et al. Anesth Analg 2007; 105:905-17; Fries et al. Anesth Analg 2002, 94:1280-7. Some recommendations quote a threshold fibrinogen concentration of 1 g/L based on the results of a study in which four out of four patients with a fibrinogen concentration of less than 0.5 g/L were seen to have diffuse microvascular bleeding. Spahn et al., Crit Care 2007, 11:R17; Stainsby et al. Br J Haematol 2006; 135:634-41; Ciavarella et al. Br J Haematol 1987; 67:365-8. By contrast, recent clinical data from peripartal hemorrhage, neurosurgery and cardiac surgery show that at a fibrinogen concentration of less than 1.5-2 g/L, there is already an increased tendency for peri- and postoperative bleeding. Charbit et al, J Thromb Haemost 2007, 5:266-73; Gerlach et al. Stroke 2002, 33:1618-23; Blome et al. J Thromb Haemost 2005, 93:1101-7; Ucar et al. Heart Surg Forum 2007, 10:E392-6.
Coagulation problems associated with bleeding disorders such as dilutional coagulopathy can be addressed with haemostatic therapy. The aim of haemostatic therapy is to minimize blood loss, transfusion requirements, and mortality. In trauma patients with identical Injury Severity Scores (ISS), mortality virtually doubles simply as a result of coagulopathy. Brohi et al. J Trauma 2003, 54:1127-30. Massive bleeding or massive transfusion in multi-traumatized patients is associated with impaired coagulation. Thus, to achieve adequate haemostasis, a sufficient amount of thrombin and sufficient coagulable substrate are required. Key elements in coagulation are the formation of thrombin on the platelet surface and the cleavage of fibrinogen by thrombin to form fibrin. Brohi et al Trauma 2003, 54:1127-30. If sufficient thrombin is formed, it converts fibrinogen to stable fibrin, which determines the firmness of the developing clot in the presence of factor XIII (FXIII). Korte, et al., Hamostaseologie 2006, 26:S30-5.
Aggressive fluid replacement is crucial in th.e case of massive blood loss, in patients with blunt and multiple trauma, to maintain normovolemia. Sperry J L, et al. J Trauma 2008; 64:9-14. However, haemodynamic stabilization by administering large amounts of crystalloid or colloid solutions causes dilutional coagulopathy. The clinical effects of impaired plasma clotting caused by normovolemic haemodilution were previously investigated in several publications. Innerhofer P et al. Anesth Analg 2002; 95:858-65, Singbartl et al, Anesth Analg 2003;96:929-35. Mittermayr et al. Anesth Analg 2007; 105:905-17.
Dilutional coagulopathy is usually treated with fresh frozen plasma (FFP) and, if available, with cryoprecipitate. Stainsby et al. Br J Anaesth 2000, 85:487-91; Spahn et al. Crit Care 2007, 11:R17. However, critically reduced clotting factor concentrations can hardly be corrected by administering FFP because of its low concentrations of coagulation factors and its volume-expanding effects, which counterbalance the intended increase in concentration of any protein of interest. Mittermayer 2007, supra; Scalea et al. Ann Surg, 2008, 248:578-84; Chowdhury et al., Br J Haematol 2004, 125:69-73; Stanworth et al. Br K Haematol 2004, 126:139-52; Abdel-Wahabet al. Transfusion 2006, 46:1279-85. In addition, administration of FFP is associated with several complications such as onset of lung injury, multiple organ failure, and infection. Watson G A, et al, J Trauma 2009, 67:221-7; Sarani et al. Crit Care Med 2008, 36:1114-8; Dara et al. Crit Care Med. 2005, 33:2667-71. Moreover, the necessary thawing process delays immediate treatment, which is of special importance in the case of acute and severe bleeding.
The effect of administration of fibrinogen concentrate on dilutional coagulopathy was previously examined in vitro, in several animal models as well as in clinical practice. Fries et al. Br J Anaesth 2005, 95:172-7; Fries et al. Anesth Analg 2006, 102:347-51; Velik-Salchner et al. J Thromb Haemost 2007, 5:1019-25; Stinger et al. J Trauma 2008, 64:S79-85. Administration of fibrinogen concentrate alone normalizes clot strength, but not initiation of coagulation, as this is a thrombin-dependent reaction. Fries et al. Br J Anaesth 2005, 95:172-7; Fries et al. Anesth Analg 2006, 102:347-51; Fenger-Eriksen et al. J Thromb Haemost 2009, 7:795-802. Thus, combinations of fibrinogen and clotting factor complexes such as pro-thrombin complex concentrate (PCC) have been previously studied.
In particular, Fries et al. studied the effect of fibrinogen substitution on reversal of dilutional coagulopathy in an in vitro model. British Journal of Anaesthesia, 2006, 97(4):460-467. Blood from 5 healthy male volunteers was diluted by 60% using lactated Ringer's solution, 4% modified gelatin solution, or 6% hydroxyethyl starch (HES) 130/0.4, as well as the combination of lactated Ringer's solution with either of 2 colloid solutions. Fries et al., Anesth Analg; 2006, 102:347-51. Thereafter, aliquots of diluted blood samples were incubated with 3 different concentrations of fibrinogen (0.75, 1.5, and 3.0 g/L). Measurements were performed by modified thrombelastography (ROTEM®; Pentapharm, Munich, Germany). After 60% dilution, clotting times increased, whereas clot firmness and fibrin polymerization decreased significantly. After administration of fibrinogen, clotting times decreased. Clot firmness, as well as fibrin polymerization, increased in all diluted blood samples when fibrinogen was added. The effect of in vitro fibrinogen substitution on ROTEM® variables was dependent on the fibrinogen dosage and the type of solution used to dilute the blood samples.
In another study, Fries et al. investigated the effect of fibrinogen and prothrombin complex concentrate (PCC) under conditions of haemodilution and uncontrolled haemorrhage in a porcine model. Fries et al., British ,Journal of Anaestesia, 2006, 97(4):460-467. After major blood loss of 65% of the estimated total blood volume, fluid volume replacement with HES (2500 ml) resulted in dilutional coagulopathy as measured by conventional coagulation tests and ROTEM® analysis. In animals receiving salvaged red blood cell concentrate only and in the placebo group, there was a small statistically significant improvement in ROTEM® parameters. However, in the treatment group, additional substitution of fibrinogen and PCC resulted in normalization of coagulation parameters. Blood loss and mortality after standardized liver injury were significantly diminished in animals treated with fibrinogen and PCC as compared with placebo.
PCC is often administered in clinical practice in the case of prolonged clotting time in critically ill patients as well as in massive bleeding situations. Schochl et al. Anaesthesia 2009. PCC corresponds of factors II, VII, IX and X as well as protein C and small amounts of heparin and has been used for years to treat hereditary coagulation deficiencies and for reversal of anticoagulation after administration of vitamin K antagonists. Only limited animal data are available on the use of modern PCC preparations in pigs exhibiting acquired coagulation factor deficiencies caused by massive blood loss and administration of HES. Dickneite et al. Anesth Analg 2008, 106:1070-7; Dickneite et al. Br J Anaesth 2009, 102:345-54; Dickneite et al J Trauma 2009.
Staudinger et al. investigated the effect of PCC on plasma coagulation in critically ill patients and pointed out that a dose of 2,000 Factor IX units of PCC (mean 30 IU/kg body weight) normalized Prothrombin Time (PT) by raising the plasma level of coagulation Factors II, VII, IX and X in patients with moderately reduced coagulation activity. Staudinger et al. intensive Care Med 1999, 25:1105-10. However, with regard to thrombin generation, PCC seems to be much more active than rhFVIIa. Dickneite et al Trauma 2009. PCC may be associated with an increased risk for thromboembolic. complication, especially in patients with acquired coagulation defects. Bagot et al. Thromb Haemost 2007, 98:1141-2; Warren et al. Ann Emerg Med 2009, 53:758-61; Kohler et al. Thromb Haemost 1998, 80:399-402.
Various additional methods for treatment of bleeding and blood loss have also been proposed, including supplementation of coagulation factors. See, for example, U.S. Patent Publications 2003/0129183, 2004/0198647, 2004/0237970, 2005/0282771, 2006/0025336, 2006/0211621, 2007/0232788, 2008/0014251, 2008/0188400, 2008/0267940, 2010/0093607, 2009/0098103, 2009/0148502, 2009/0175931, 2009/0232877, and 2010/0086529.
Despite the proposal and study of various compositions and methods for restoring haemostasis, there remains a need in the art for improved methods of treating haemostatic disorders such as dilutional coagulopathy.
To achieve haemostasis in patients with bleeding disorders or blood loss, a sufficient amount of thrombin and sufficient coagulable substrate (fibrinogen) are required. Key aspects in coagulation are the formation of thrombin on the platelet surface and the cleavage of fibrinogen by thrombin to form fibrin. If sufficient thrombin is formed, it converts fibrinogen to stable fibrin provided enough fibrinogen is present, which determines the firmness of the developing clot in the presence of factor XIII (FXIII).
The present invention provides for the treatment or minimization of uncontrolled bleeding using recombinant haemostatic agent(s), including recombinant human factor II FII) alone or a combination of three recombinant human coagulation factors, rhII, rhFX and rhVIIa (3F), were the haemostatic agents are used with or without fibrinogen. In particular, the present invention demonstrates the efficacy of such recombinant haemostatic agents in the treatment of dilutional coagulopathy. Surprisingly, administration of rhFII alone or in combination with fibrinogen is sufficient to restore normal haemostasis. Administration of rhFII alone, or in combination with fibrinogen, can avoid the potentially serious complications of thromboembolic events that may accompany other tandard forms of ent currently provided to control bleeding disorders or blood loss.
It should be noted that reference to any publications herein should not be construed as an admission that such a publication is prior art to the inventions disclosed herein below.